Development of multitargeted inhibitors of both the insulin-like growth factor receptor (IGF-IR) and members of the epidermal growth factor family of receptor tyrosine kinases

Article abstract of DOI:10.1016/j.bmcl.2009.01.086

Emerging clinical and pre-clinical data indicate that both insulin-like growth factor receptor (IGF-IR) and members of the epidermal growth factor (EGF) family of receptor tyrosine kinases (RTKs) exhibit significant cross-talk in human cancers. Therefore, a small molecule that successfully inhibits the signaling of both classes of oncogenic kinases might provide an attractive agent for chemotherapeutic use. Herein, we disclose the structure activity relationships that led to the synthesis and biological characterization of 14, a novel small molecule inhibitor of both IGF-IR and members of the epidermal growth factor family of RTKs.

Full text of DOI:10.1016/j.bmcl.2009.01.086

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1718–1721
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of multitargeted inhibitors of both the insulin-like growth
factor receptor (IGF-IR) and members of the epidermal growth factor
family of receptor tyrosine kinases
*
Robert D. Hubbard , Nwe Y. Bamaung, Steve D. Fidanze, Scott A. Erickson, Fabio Palazzo, Julie L. Wilsbacher,
Qian Zhang, Lora A. Tucker, Xiaoming Hu, Peter Kovar, Donald J. Osterling, Eric F. Johnson, Jennifer Bouska,
Jieyi Wang, Steven K. Davidsen, Randy L. Bell, George S. Sheppard
Cancer Research, Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Emerging clinical and pre-clinical data indicate that both insulin-like growth factor receptor (IGF-IR) and
members of the epidermal growth factor (EGF) family of receptor tyrosine kinases (RTKs) exhibit signif-
icant cross-talk in human cancers. Therefore, a small molecule that successfully inhibits the signaling of
both classes of oncogenic kinases might provide an attractive agent for chemotherapeutic use. Herein, we
disclose the structure activity relationships that led to the synthesis and biological characterization of 14,
a novel small molecule inhibitor of both IGF-IR and members of the epidermal growth factor family of
RTKs.
Received 11 December 2008
Revised 26 January 2009
Accepted 27 January 2009
Available online 30 January 2009
Keywords:
Insulin-like growth factor receptor (IGF-IR)
EGFR
Ó 2009 Elsevier Ltd. All rights reserved.
ErbB-2
Kinase
Inhibitor
Pyrazolo[3,4-d]pyrimidine
The development and maintenance of cancer requires dysregu-
lation of normal cellular signaling. Receptor tyrosine kinases serve
to propagate external cellular stimuli into intracellular responses
that range from proliferation, to induction of cell death. In a variety
of cancers, RTKs are either mutated or overexpressed leading to
enhanced proliferation, resistance to chemotherapy or decreased
apoptotic potential. Therefore, several small molecule inhibitors
have been developed that inhibit RTKs, such as Bcr-Abl (GleevecÒ),
and KDR (NexavarÒ).1 For the agents mentioned above, these drugs
inhibit multiple kinase targets, potentially enabling broader clini-
cal utility versus a more selective agent.2 A key issue in developing
the next generation of RTK inhibitors is to determine which kinases
should be targeted by a single small molecule; one potential com-
bination is IGF-IR and members of the EGF family of RTKs.
IGF-IR is a member of a complex system of growth factors and
receptors that includes: insulin receptor (IR), insulin-like growth
factor I/II (IGF-I/IGF-II), and six insulin-like growth factor binding
proteins.3 In normal tissues, IGF-IR is essential for growth, develop-
ment, and the suppression of apoptosis. This is a consequence of
the ability of IGF-IR to simultaneously activate the anti-apoptotic
phosphoinositide-3-kinase/Akt pathway, and the mitogenic
extracellular signal regulated kinase (ERK)/mitogen-activated
protein kinase pathway (MAPK).4 In malignant cells, the ability of
IGF-IR to simultaneously control both the pro-survival and the pro-
liferative aspects of the cellular machinery allow these cells to
avoid apoptosis, induce the production of key angiogenic factors,
and promote tumor cell invasion.5
The epidermal growth factor family of RTKs consists of four
family members: the epidermal growth factor receptor (EGFR/
ErbB-1), ErbB-2, ErbB-3 and ErbB-4. ErbB-2 is an orphan receptor
and ErbB-3 lacks kinase activity. Normally, the ErbB-family mem-
bers bind growth factor then proceed to either homodimerize or
heterodimerize leading to phosphorylation and activation of the
receptor pair.6 However, receptor overexpression of ErbB-2, via
gene amplification, can drive ligand independent activation. For
EGFR, aberrant activation can be achieved either via autocrine
overexpression of its ligands,7 or by possessing activating muta-
tions in and around the kinase domain of the receptor.8 Analogous
to IGF-IR, the ErbB-family of RTKs signal through both MAPK and
Akt pathways, thereby enabling the receptors to control various
cellular processes including proliferation, migration and evasion
of apoptosis.
Recent literature has suggested that simultaneous inhibition of
IGF-IR and EGFR/ErbB-2 with combinations of either small
molecules or antibodies affords enhanced inhibition of cellular
* Corresponding author. Tel.: +1 847 936 9242; fax: +1 847 935 5165.
E-mail address: robert.d.hubbard@abbott.com (R.D. Hubbard).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.086

R. D. Hubbard et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1718–1721
1719
Table 1
NH2
O
O
NH2
NO2
Enzyme activities of substituted benzimidazoles
c
a, b
B
+
O
R
B
NH2
N
2
Br
O
1
2
NH
3
NH2
N
N
N
R
O
N
H
N
B
N
O
4
R
N
NH
N
NH2
N
I
NH2
N
N
O
N
N
d, e
N
N
N
Analog
R=
Enzyme (IC50, nM)
EGFRa
IGF-IR
64
ErbB-2
186
6
7
270
361
O
R2
6-15
N
R1
5
25
36
250
664
S
Scheme 1. Reagents and conditions: (a) PdCl2ꢀdppf, KOAc, DMF, 100 °C, 85–99%; (b)
10% Pd/C, 60 psi H2, EtOAc, 91%; (c) 1—CDI, THF, R-CO2H; 2—3, AcOH, 90 °C, 60–
70%; (d) amine (R1R2NH), HCO2H, NMP, 100 °C, 25% yield of trans-diastereomer; (e)
4, (Ph3P)2PdCl2, 2 M aq Na2CO3, DME/H2O 2/1 (v/v), 80 °C, 40–87%.
Cl
8
65
58
proliferation versus the single agents. In particular, the combina-
tion of IGF-IR and EGFR antibodies showed improved activity ver-
sus an A549 mouse tumor xenograft.9 Combinations of gefitinib
and NVP-ADW742 (selective EGFR and IGF-IR inhibitors, respec-
tively) caused synergistic decreases in cellular proliferation across
a diverse set of cancer cell lines.10 The impressive combination
data provides rationale for a small molecule discovery program
targeting simultaneous inhibition of IGF-IR and EGFR/ErbB-2. We
have previously disclosed that a subset of pyrazolo[3,4-d]pyrimi-
dines possesses potent IGF-IR inhibitory properties,11 however,
these analogs displayed poor cellular activity versus EGFR/ErbB-
2. Therefore, the synthetic efforts were focused on the preparation
of a scaffold that provided balanced enzyme and cellular activities
versus the three targets of interest: IGF-IR, EGFR, and ErbB-2.
The synthesis of the pyrazolopyrimidine analogs begins with
boronation of the commercially available bromide, 2, and subse-
quent reduction of the nitro-arene intermediate affords 3 in 77–
80% yield over the two steps (Scheme 1). Acylation of the desired
phenylacetic-acid derivative with 1,10-carbonyldiimidazole (CDI)
yields the crude acyl-imidazole, which was treated with 3 in the
presence of acetic acid12 to afford the benzimidazole 4 in 60–70%
yield. Reductive amination of the known ketone 5,13 followed by
Suzuki coupling with 4 affords the fully elaborated set of analogs
(6–15).
Previous SAR indicated that a trans-substituted morpholino-
pyrazolopyrimidine afforded potent IGF-IR inhibition.14 With the
amine held constant, we assessed the effect of benzimidazole sub-
stitutions on EGFR/ErbB-2 enzyme potency (Table 1).15 The simple
phenyl (6) and the isosteric thiophene (7) analogs yielded potent
enzyme inhibition for IGF-IR, but only moderate activity versus
the ErbB-family members. The 2-chloro substituent, in 8, which
was previously reported to provide IGF-IR activity,11 demonstrated
increased potency versus EGFR, but not against ErbB-2. Changing
from 2-Cl to 2-OMe yielded 9, a compound that possessed balanced
enzyme inhibition across the desired targets.
O
9
81
53
a
EGFR kinase construct possessed the L858R activating mutation.
examining the kinase activity of the compounds, analogs were
assessed for their ability to inhibit cellular phosphorylation in rel-
evant cancer cell lines: MiaPaCa-2, which expresses both IGF-IR
and EGFR and N87, which expresses ErbB-2.16 The simple pipera-
zine 10, afforded balanced enzyme activity, however, suffered from
poor cellular activity. A survey of nitrogen-based functional groups
(amide, carbonate, and urea) was prepared to tune the basicity of
the piperazinyl-nitrogen yielding analogs (11, 12, and 13). Collec-
tively, this set of analogs demonstrated poor cellular activity. How-
ever, an alkyl-substituted piperazine 14 displayed the desired
balance of enzyme and cellular activity required for further evalu-
ation. Although the enzyme and cellular activity for 15 were com-
parable to 14, phamacokinetic evaluation of 15 in mice resulted in
much lower oral exposure (data not shown).
The ADME and pharmacokinetic (PK) properties of 14 are sum-
marized in Figure 1. The in vitro ADME characterization of 14
reveals that the compound possesses relatively low plasma protein
binding and very high aqueous solubility. In addition, analog 14
displays a fairly benign cytochrome P450 inhibition profile. The
murine PK of 14 shows low clearance coupled with moderate oral
bioavailability, which increases via IP administration (12% vs
>100%).
The balanced kinase and cellular activity of 14 coupled with the
moderate murine PK indicated that this compound warranted fur-
ther investigation in our in vivo phamacodynamic (PD) model. The
experimental design of the PD model is such that the compound is
dosed either IP or PO and the mice (N = 3) are challenged 4 h later
with growth factor stimulation, via an IV dose of IGF-I and EGF. The
level of inhibition is determined by examining the phosphorylation
status of the receptor in either lung (IGF-IR) or liver (EGFR). As
shown in Figure 2, analog 14 dosed via either IP or PO (50 mg/
With the preferred benzimidazole substituent (2-OMe) identi-
fied, we focused on varying the amine moiety. The results from
the amine-group SAR are shown in Table 2. In addition to

1720
R. D. Hubbard et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1718–1721
Table 2
Enzyme and cellular activity of 2-OMe substituted pyrazolopyrimidines
O
N
NH
NH2
N
N
N
N
R
Analog
R=
Enzyme (IC50, nM)
EGFRa
Cellular phosphorylation (EC50, nM)
IGF-IR
26
ErbB-2
44
IGF-IR (MiaPaCa-2)
EGFR (MiaPaCa-2)
ErbB-2 (N87)
210
10
30
130
30
25
58
3
711
143
247
208
115
190
335
N
N H
O
11
12
13
14
27
309
69
59
270
17
742
520
808
85
660
1600
1580
94
N
N
N
N
N
N
O
O
O
N
81
54
N
N
N
N
O
15
40
13
91
147
a
EGFR kinase construct possessed the L858R mutation.
In vitro ADME
P450 inhibition Profile
IGF1/EGF (0.5 mg/kg, iv)
A
CYP2C9
CYP3A4
CYP2D6
pH = 7.2
IC50 > 20 µM
IC50 > 20 µM
IC50 = 1.6 µM
> 30 µM
14 @ 50 mg/kg
BK
Veh, PO
PO
IP
Aqueous Solubility
Human Protein Binding
96.7%
-
pIGF IR
Mouse PK
PO DNAUCa
IV DNAUC
IP DNAUC
F (PO)
Units
-
IGF IR
0.42
3.50
2.71
12
> 100
0.47
3.1
µmol·hr/L/mg/kg
µmol·hr/L/mg/kg
µmol·hr/L/mg/kg
%
%
IGF1/EGF (0.5 mg/kg, iv)
B
F (IP)
Cl
t1/2
@ 50 mg/kg
PO IP
14
L/hr/kg
hr
a. DNAUC = dose normalized AUC
BK
Veh, PO
pEGFR
EGFR
Figure 1. ADME and murine PK characterization of 14.
kg) resulted in near complete inhibition of IGF-IR and EGFR kinase
activity (receptor phosphorylation).
Figure 2. In vivo activity of 14 in a murine PD model of efficacy. (A) Represents the
inhibition of lung IGF-IR phosphorylation after an IP or PO dose of 14 (BK = back-
ground). (B) Represents the inhibition of liver EGFR phosphorylation after an IP or
PO dose of 14 (BK = background).
In conclusion, we have disclosed that appropriate functionaliza-
tion of a novel pyrazolopyrimidine scaffold yields a series of mult-
itargeted kinase inhibitors. In particular, analog 14 displayed a
balanced enzyme and cellular inhibitory profile versus IGF-IR and
members of the epidermal growth factor RTK family (EGFR and
ErbB-2). The in vitro activity parlayed into in vivo activity as deter-
mined in a murine PD model, in which 14 completely inhibited

R. D. Hubbard et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1718–1721
1721
11. Hubbard, R. D.; Bamaung, N. Y.; Palazzo, F.; Zhang, Q.; Kovar, P.; Osterling, D. J.;
Hu, X.; Wilsbahcer, J. L.; Johnson, E. F.; Bouska, J.; Wang, J.; Davidsen, S. K.;
Sheppard, G. S. Bioorg. Med. Chem. Lett. 2007, 17, 5406.
receptor phosphorylation of both IGF-IR and EGFR. Additional
in vivo characterization of 14 will be disclosed in due course.
12. Lin, R.; Chiu, G.; Yu, Y.; Connolly, P. J.; Li, S.; Lu, Y.; Adams, M.; Feuntes-
Pesquera, A. R.; Emanuel, S. L.; Greenberger, L. M. Bioorg. Med. Chem. Lett. 2007,
17, 4557.
References and notes
13. Wishart, N.; Friedman, M.; Arnold, L. D.; Yang, B.; Fix-Stenzel, S. R.; Ericsson, A.;
Michaelides, M. R.; Qian, X.-D.; Holmes, J. H.; Steinman, D. H.; Tian, Z.;
Wittenberger, S. J. PCT Int. Appl. 2005. WO20005074603.
14. This class of compounds possesses equivalent enzyme and cellular potency for
both IGF-IR and IR.
15. The kinase inhibitory activity of the disclosed compounds were assayed by a
homogeneous time-resolved fluorescence assay as previously described, see
Ref. 11 The IC50s for IGF-IR (0.25 nM, Cell Signaling Technology) and EGFR–
1. (a) Arora, A.; Scholar, E. M. J. Pharmacol. Exp. Ther. 2005, 315, 971; (b) Tibes, R.;
Trent, J.; Kurzrock, R. Annu. Rev. Pharmacol. Toxicol. 2005, 45, 357.
2. Daub, H.; Specht, K.; Ullrich, A. Nat. Rev. Drug Disc. 2004, 3, 1001.
3. Foulstone, E.; Prince, S.; Zaccheo, O.; Burns, J. L.; Harper, J.; Jacobs, C.; Church,
D.; Hassan, A. B. J. Pathol. 2005, 205, 145.
4. Yakar, S.; LeRoith, D.; Brodt, P. Cytokine Growth Factor Recept. Rev. 2005, 16, 407.
5. (a) Wang, Y.; Sun, Y. Curr. Cancer Drug Targets 2002, 2, 191; (b) Burtscher, I.;
Christofori, G. Drug Resist. Updat. 1999, 2, 3.
6. Wieduwilt, M. J.; Moasser, M. M. Cell. Mol. Life Sci. 2008, 65, 1566.
7. Hynes, N. E.; Lane, H. A. Nat. Rev. Cancer 2005, 5, 341.
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N. Int. J. Cancer 2005, 113, 316.
L858R (4 nM, Upstate) were obtained using 50
lM
and 100
lM
ATP,
respectively.
16. MiaPaCa-2 and N87 cells were treated with serial dilutions (0.0003–30
lM) of
compounds for 6 h in growth media containing 10% fetal bovine serum.
MiaPaCa-2 cells were then stimulated with IGF-I and EGF for 10 min. Cells
were lysed and phosphorylation of IGF-IR, EGFR, and ErbB-2 was determined
using the human p-IGF-IR, human p-EGFR, and human p-ErbB-2 DuoSet IC
ELISA kits from R&D Systems (Minneapolis, MN).
10. Wilsbacher, J. L.; Zhang, Q.; Tucker, L. A.; Hubbard, R. D.; Sheppard, G. S.;
Bamaung, N. Y.; Fidanze, S. D.; Wang, G. T.; Hu, X.; Davidsen, S. K.; Bell, R. L.;
Wang, J. J. Biol. Chem. 2008, 283, 23721.